Correlation Between Expression of Recombinant Proteins and Abundance of H3K4Me3 on the Enhancer of Human Cytomegalovirus Major Immediate-Early Promoter

Abstract

Role of epigenetic regulation in the control of gene expression is well established. The impact of several epigenetic mechanisms, such as DNA methylation and histone acetylation, on recombinant protein production in mammalian cells has been investigated recently. Here we investigate the correlation between the selected epigenetic markers and five trastuzumab biosimilar-producing Chinese hamster ovary (CHO) cell lines in which the expression of trastuzumab is driven by human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. We chose the producing clones in which transcription was the determinative step for the production of recombinant trastuzumab. We found that the abundance of trimethylation of histone 3 at lysine 4 (H3K4Me3) on the enhancer of HCMV MIE promoter correlated well with the relative titers of recombinant trastuzumab among the clones. Such close correlation was not observed between the recombinant protein and other epigenetic markers examined in our study. Our results demonstrate that the HCMV MIE enhancer-bound H3K4Me3 epigenetic marker may be used as the epigenetic indicator to predict the relative production of recombinant proteins between the producing CHO cell lines.

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

“…Numerous studies have shown correlations between activating or repressive histone modifications associated with the major IE gene and the levels of viral gene expression. For example, association of the MIEP with H3K4me2, H3K4me3, H3K9/14ac, H3S10ph or H4Kac has been linked to high levels of IE (or transgene) transcription and productive infection or reactivation from latency [72,[110][111][112][113][114][115][116][117][118][119][120]. By contrast, the presence of H3K9me2, H3K9me3 or H3K27me3 at the MIEP generally correlates with low levels of IE transcription and either latent or the onset (pre-IE phase) of productive infection [110][111][112]115,116] ( Figure 1).…”

Regulation of the MIE Locus During HCMV Latency and Reactivation

“…Methylation of H3K4 and phosphorylation of serine 10 of H3 (H3S10ph) are also characteristic of transcriptional activation [154,155]. Thus, it is unsurprising that H3K4me2, H3K4me3, H3K9/14ac, H3S10ph, and H4Kac recruitment to the MIE locus results in increased gene transcription from this region ( Figure 2) [48,49,[131][132][133][134][135]156,157], suggesting these modifications render this enhancer/promoter more accessible to transcription factors that promote active transcription. Histone acetyltransferases (HATs; e.g., KAT6A/MOZ) and demethylases (e.g., KDM1A/LSD1, KDM4A/JMJD2, KDM6B/JMJD3) activate the MIE locus by adding acetyl groups and removing methyl groups from histones at the MIE enhancer region [140], which is necessary for transcription initiation from this locus during reactivation.…”

Key Challenges in Designing CHO Chassis Platforms

“…To avoid this, genetic sequences that maintain an open chromatin configuration have been used [195,196]. Direct silencing of the CMV promoter used for most transgenes, either by DNA-methylation or by changes in the histone modifications has also been shown [197][198][199][200]. These different strategies to silence the gene of interest are most likely due to the cell's drive to remove the stress of high productivity [201][202][203], by which ever means possible.…”